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This chapter provides an overview on current methods applied for the study of the microbiome from environmental samples (e.g., forest trees). The study of microbiomes was traditionally involving culture-dependent methods for isolation and characterization of the microbes. A major constraint with culture-based methods is that many unculturable microbes cannot be isolated or profiled. The application of high-throughput sequencing (HTS) together with advances in bioinformatics has facilitated culture-independent analyses of the tree microbiome. Other “omics” approach such as RNA-seq has recently been applied for taxonomic classification, as it has additional advantage of identifying transcriptionally active microbial communities. The genomics methods can be further complemented with metabolomics and metaproteomics approaches for the profiling of microbiota communities. The decreasing cost of HTS has greatly enabled efforts to decipher the microbiota community of forest biomes.

The tree phyllosphere, endosphere, and rhizosphere microbiota is a complex ecosystem with several diverse functions that form an integral part of the host metabolism. Despite the perceived progress in microbiome study, the compositions of tree microbiota (viruses, archaea, bacteria, and fungi) community are still not fully uncovered. The application of “omics” technology including next generation or high throughput sequencing (NGS, HTS) and metagenomics could help to facilitate in unraveling the ecological community of tree microbiota. These technological advances are needed to expand the opportunity to gain fresh insights that will facilitate a better understanding on the functional role of the microbiota in tree health and disease progression. Linking results of microbiome study to practical applications, however, currently remains a major challenge in forest protection. In the long term, unraveling the microbiome of forest biome has the potential to expedite improved forest management as well as sustainable disease control strategies.

15 p.-6 fig.-3 tab., Fungal plant pathogens remain a serious threat to the sustainable agriculture and forestry, despite the extensive efforts undertaken to control their spread. White root rot disease is threatening rubber tree (Hevea brasiliensis) plantations throughout South and Southeast Asia and Western Africa, causing tree mortality and severe yield losses. Here, we report the complete genome sequence of the basidiomycete fungus Rigidoporus microporus, a causative agent of the disease. Our phylogenetic analysis confirmed the position of R. microporus among the members of Hymenochaetales, an understudied group of basidiomycetes. Our analysis further identified pathogen’s genes with a predicted role in the decay of plant cell wall polymers, in the utilization of latex components and in interspecific interactions between the pathogen and other fungi. We also detected putative horizontal gene transfer events in the genome of R. microporus. The reported first genome sequence of a tropical rubber tree pathogen R. microporus should contribute to the better understanding of how the fungus is able to facilitate wood decay and nutrient cycling as well as tolerate latex and utilize resinous extractives., This project was financially supported by Academy of Finland (Grant No. 307580). The work conducted by the U.S. Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, is supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. A.T.M., S.C., F.J.R.D. and A.S. were funded by the EnzOx2 (BBI-PPP-2015-2-720297; www.enzox2.eu) EU project, and the GENOBIOREF (BIO2017-86559-R) project of the Spanish Ministry of Economy, Industry and Competitiveness,co-financed by FEDER funds.

Fall armyworm (FAW),Spodoptera frugiperda, is an invasive insect pest with a diverse host range threatening food security in the African continent by causing severe economic damage to staple maize crop. FAW like all other insects are associated with various microbiota that influence their several characteristics and activities. The microbiota of insects plays important roles in their growth, development and environmental adaptation to their host plants or animals and not much is known about microbiota of FAW in Nigeria. In this study, the microbial community of FAW larvae from maize plants in Nigeria was investigated using Illumina PE250 NovaSeq of 16S rRNA V3-V4 gene region. A total of 1,160,133 sequence reads that ranged from 83,794 to 145,475 were obtained. They span through 2 kingdoms: bacteria (99.59%) and archaea (0.37%), 37 phyla, 59 classes, 78 orders, 145 families and 285 genera. The dominant taxonomic group of bacteria across the larval samples were Proteobacteria (58.32%), Firmicutes (35.87%) and Bacteroidetes (4.02%). There were no significant differences in bacteria species diversity and richness among the individual fall armyworm larva and samples from different geographical regions. The relative abundance of Bacteria phyla and classes also showed no significant differences among the larval samples. Significant differences were documented in bacteria orders Acidobacteriales and Aeromonadales, family Acidobacteriaceae_ (Subgroup_1) and genera Novosphingobium and Pedobacter. The results indicate that the gut of FAW larvae harbours a diverse set of bacteria and archaea biota. Understanding their ecological and functional relevance would provide novel insight on potential approaches for their exploitation in the integrated management of FAW in the tropics.

In the forest of Northern Hemisphere, the fungi Heterobasidion annosum (Fr.) Bref. s.l. causes severe root and stem rot diseases, dramatically reducing the wood quality of conifer trees. The hallmark of the host response during the infection process is the formation of necrotic lesions and reaction zones. To characterize physiochemical and molecular features of the necrotic lesion, we conducted artificial inoculations on Norway spruce plants at different developmental stages: seedlings, young and mature trees. The results were further compared against data available on the formation of reaction zones. Strong necrosis browning or enlarged necrotic lesions were observed in infected tissues. This was accompanied by elevated pH. However, the increased pH, around 6.0 in necrotic lesions, was not as high as that documented in reaction zones, above 7.0 as marked by the intensity of the blue colour in response to 2,6-dichlorophenol indophenol dye. Peroxidase activity increased in infected plants and RNA-seq analysis of necrotic lesions showed marked upregulation of defence-related genes. Our findings highlight similarities and differences between the reaction zone and necrotic lesion formation in response of conifer trees to biotic stress.

Pine wilt disease (PWD) caused by the nematode Bursaphelenchus xylophilus is a devastating disease in conifer forests in Eurasia. However, information on the effect of PWD on the host microbial community is limited. In this study, the bacterial community structure and potential function in the needles, roots, and soil of diseased pine were studied under field conditions using Illumina MiSeq coupled with Phylogenetic Investigation of Communities by Reconstruction of Unobserved states (PICRUSt) software. The results showed that the community and functional structure of healthy and diseased trees differed only in the roots and needles, respectively (p &lt, 0.05). The needles, roots, and soil formed unique bacterial community and functional structures. The abundant phyla across all samples were Proteobacteria (41.9% of total sequence), Actinobacteria (29.0%), Acidobacteria (12.2%), Bacteroidetes (4.8%), and Planctomycetes (2.1%). The bacterial community in the healthy roots was dominated by Acidobacteria, Planctomycetes, and Rhizobiales, whereas in the diseased roots, Proteobacteria, Firmicutes, and Burkholderiales were dominant. Functionally, groups involved in the cell process and genetic information processing had a higher abundance in the diseased needles, which contributed to the difference in functional structure. The results indicate that PWD can only affect the host bacteria community structure and function in certain anatomical regions of the host tree.

Plant microbiome plays an important role in maintaining the host fitness. Despite a significant progress in our understanding of the plant microbiome achieved in the recent years, very little is known about the effect of plant pathogens on composition of microbial communities associated with trees. In this study, we analysed the mycobiome of different anatomic parts of asymptomatic and symptomatic Norway spruce trees naturally infected by Heterobasidion spp. We also investigated the primary impact of the disease on the fungal communities, which are associated with Norway spruce trees. Our results demonstrate that the structure of fungal communities residing in the wood differed significantly among symptomatic and asymptomatic Heterobasidion-infected trees. However, no significant differences were found in the other anatomic regions of the trees. The results also show that not only each of individual tree tissues (wood, bark, needles and roots) harbours a unique fungal community, but also that symptomatic trees were more susceptible to co-infection by other wood-degrading fungi compared to the asymptomatic ones.

The causal agent of root and butt rot of conifer trees, Heterobasidion annosum, is widespread in boreal forests and economically responsible for annual loss of approximately 50 million euros to forest industries in Finland alone and much more at European level. In order to further understand the pathobiology of this fungus at the genome level, a Finnish isolate of H. annosum sensu stricto (isolate 03012) was sequenced and analyzed with the genome sequences of 23 white-rot and 13 brown-rot fungi. The draft genome assembly of H. annosum has a size of 31.01 Mb, containing 11,453 predicted genes. Whole genome alignment showed that 84.38% of H. annosum genome sequences were aligned with those of previously sequenced H. irregulare TC 32-1 counterparts. The result is further supported by the protein sequence clustering analysis which revealed that the two genomes share 6719 out of 8647 clusters. When sequencing reads of H. annosum were aligned against the genome sequences of H. irregulare, six single nucleotide polymorphisms were found in every 1 kb, on average. In addition, 98.68% of SNPs were found to be homo-variants, suggesting that the two species have long evolved from different niches. Gene family analysis revealed that most of the white-rot fungi investigated had more gene families involved in lignin degradation or modification, including laccases and peroxidase. Comparative analysis of the two Heterobasidion spp. as well as white-/brown-rot fungi would provide new insights for understanding the pathobiology of the conifer tree pathogen.

The basidiomycete Heterobasidion annosum sensu lato (s.l.) is considered to be one of the most destructive conifer pathogens in the temperate forests of the northern hemisphere. H. annosum is characterized by a dual fungal lifestyle. The fungus grows necrotrophically on living plant cells and saprotrophically on dead wood material. In this study, we screened the H. annosum genome for small secreted proteins (HaSSPs) that could potentially be involved in promoting necrotrophic growth during the fungal infection process. The final list included 58 HaSSPs that lacked predictable protein domains. The transient expression of HaSSP encoding genes revealed the ability of 8 HaSSPs to induce cell chlorosis and cell death in Nicotiana benthamiana. In particular, one protein (HaSSP30) could induce a rapid, strong, and consistent cell death within 2 days post-infiltration. HaSSP30 also increased the transcription of host-defence-related genes in N. benthamiana, which suggested a necrotrophic-specific immune response. This is the first line of evidence demonstrating that the H. annosum genome encodes HaSSPs with the capability to induce plant cell death in a non-host plant.

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The initial stage of cellulose degradation has been studied via in vitro assays of fungi isolated from rotten wood in a boreal forest. Among the 37 isolates, Antrodia sinuosa appeared to be an effective cellulose degrader and was selected for studying the initial degradation process. In the liquid cultivation with carboxymethylcellulose (CMC), the increase of the mycelial dry weight coincided with the pH decrease of the culture medium from pH 5.7 to 3.9, between the 3rd and 6th cultivation day. At the same time, the cellulolytic activity increased; the CMCase activity increased sharply and the reducing sugars reached their maximum concentration in the culture medium. It seems that the decreasing pH enables the cellulose degradation by A. sinuosa at an early stage of the process. The results of this study may be useful for a more efficient industrial application of biomass by means of brown-rot fungi.

Silver birch (Betula pendula) is a pioneer boreal tree that can be induced to flower within 1 year. Its rapid life cycle, small (440-Mb) genome, and advanced germplasm resources make birch an attractive model for forest biotechnology. We assembled and chromosomally anchored the nuclear genome of an inbred B. pendula individual. Gene duplicates from the paleohexaploid event were enriched for transcriptional regulation, whereas tandem duplicates were overrepresented by environmental responses. Population resequencing of 80 individuals showed effective population size crashes at major points of climatic upheaval. Selective sweeps were enriched among polyploid duplicates encoding key developmental and physiological triggering functions, suggesting that local adaptation has tuned the timing of and cross-talk between fundamental plant processes. Variation around the tightly-linked light response genes PHYC and FRS10 correlated with latitude and longitude and temperature, and with precipitation for PHYC. Similar associations characterized the growth-promoting cytokinin response regulator ARR1, and the wood development genes KAK and MED5A.

Main conclusion Two terpene compounds and four genes were identified as potential biomarkers for further evaluation for Scots pine susceptibility or tolerance against Heterobasidion annosum. Scots pine (Pinus sylvestris) is one of the main sources of timber in the boreal zone of Eurasia. Commercial pine plantations are vulnerable to root and butt rot disease caused by the fungus Heterobasidion annosum. The pathogen affects host growth rate, causes higher mortality and decreases in timber quality, resulting in considerable economic losses to forest owners. Genetic and biochemical factors contributing to Scots pine tolerance against H. annosum infection are not well understood. We assessed the predictive values of a set of potential genetic and chemical markers in a field experiment. We determined the expression levels of 25 genes and the concentrations of 36 terpenoid compounds in needles of 16 Scots pine trees randomly selected from a natural population prior to artificial infection. Stems of the same trees were artificially inoculated with H. annosum, and the length of necrotic lesions was documented 5 months post inoculation. Higher expression level of four genes included in our analysis and encoding predicted alpha-pinene synthase (two genes), geranyl diphosphate synthase (GPPS), and metacaspase 5 (MC5), could be associated with trees exhibiting increased levels of necrotic lesion formation in response to fungal inoculation. In contrast, concentrations of two terpenoid compounds, beta-caryophyllene and alpha-humulene, showed significant negative correlations with the lesion size. Further studies with larger sample size will help to elucidate new biomarkers or clarify the potential of the evaluated markers for use in Scots pine disease resistance breeding programs.

Background The white rot fungus Phlebia radiata, a type species of the genus Phlebia, is an efficient decomposer of plant cell wall polysaccharides, modifier of softwood and hardwood lignin, and is able to produce ethanol from various waste lignocellulose substrates. Thus, P. radiata is a promising organism for biotechnological applications aiming at sustainable utilization of plant biomass. Here we report the genome sequence of P. radiata isolate 79 originally isolated from decayed alder wood in South Finland. To better understand the evolution of wood decay mechanisms in this fungus and the Polyporales phlebioid clade, gene content and clustering of genes encoding specific carbohydrate-active enzymes (CAZymes) in seven closely related fungal species was investigated. In addition, other genes encoding proteins reflecting the fungal lifestyle including peptidases, transporters, small secreted proteins and genes involved in secondary metabolism were identified in the genome assembly of P. radiata. Results The PACBio sequenced nuclear genome of P. radiata was assembled to 93 contigs with 72X sequencing coverage and annotated, revealing a dense genome of 40.4 Mbp with approximately 14 082 predicted protein-coding genes. According to functional annotation, the genome harbors 209 glycoside hydrolase, 27 carbohydrate esterase, 8 polysaccharide lyase, and over 70 auxiliary redox enzyme-encoding genes. Comparisons with the genomes of other phlebioid fungi revealed shared and specific properties among the species with seemingly similar saprobic wood-decay lifestyles. Clustering of especially GH10 and AA9 enzyme-encoding genes according to genomic localization was discovered to be conserved among the phlebioid species. In P. radiata genome, a rich repertoire of genes involved in the production of secondary metabolites was recognized. In addition, 49 genes encoding predicted ABC proteins were identified in P. radiata genome together with 336 genes encoding peptidases, and 430 genes encoding small secreted proteins. Conclusions The genome assembly of P. radiata contains wide array of carbohydrate polymer attacking CAZyme and oxidoreductase genes in a composition identifiable for phlebioid white rot lifestyle in wood decomposition, and may thus serve as reference for further studies. Comparative genomics also contributed to enlightening fungal decay mechanisms in conversion and cycling of recalcitrant organic carbon in the forest ecosystems. Electronic supplementary material The online version of this article (10.1186/s12864-019-5817-8) contains supplementary material, which is available to authorized users.

Fungal endophytes have been reported to inhibit growth of phytopathogenic microbes and some also are known to promote root growth. If endophytes of conifers can protect their host roots against invaders the screening of endophytes for their biocontrol abilities is relevant. The aim of this research was to screen for the potential inhibitory effects of selected Norway spruce root endophytes during interaction, in vitro, with well-known genera of phytopathogens (Heterobasidion parviporum, Phytophtora pini, Botrytis cinerea) and test the endophytes ability to protect Norway spruce seedlings against H. parviporum infection. The root endophytes and their metabolites were able to form inhibition zones in paired cultures with the pathogens. Higher numbers of unique metabolites were observed in culture extracts of Cryptosporiopsis sp. further suggesting that stronger inhibitory effect observed could be due to acquisition of more diverse metabolite pool. However, this endophyte decreased and retarded the root growth of Norway spruce seedlings. The endophyte Phialocephala sphareoides was able to inhibit all phytopathogens, promoted the root shoot ratio of Norway spruce seedlings and was able to prevent under in vitro conditions the infections of seedling roots by the pathogen H. parviporum.

Summary Gene expression levels of pathogenesis-related proteins (PR1, PR3, PR5, PR8 and PR9), cell wall modification genes (PAL and expansin), signal transduction genes (ACC oxidase, AOC, MAPK) and a Myb transcription factor were compared in two Hevea brasiliensis budded clones (RRIM612 and PR107) 5 weeks after wounding and artificial inoculation with the white rot fungus, Rigidoporus microporus. PR3 class I chitinase was upregulated in RRIM612 in response to the pathogen when compared with the wounded control. PR9 class IV peroxidase was highly upregulated in PR107. PR1 and PR8 were expressed more in wounded than in inoculated clones. Genes involved in ethylene and jasmonic acid signalling pathways and phenylalanine ammonia-lyase (PAL) were upregulated almost equally in both clones. The Myb transcription factor was upregulated in RRIM612, while MAPK was upregulated in PR107 when compared to the wounded control. The predicted expansin-like protein was upregulated 40-fold in RRIM612 when compared to the wounded control. The results demonstrate the variability in defence responses in different clones and provide the first set of defence genes expression profiles in the host–pathogen interaction of the white rot disease of rubber trees.

Background and objectives: The global forest economy is threatened by eucalypt pathogens which are often latent or cryptic species that escape common quarantine and detection methods. Plantation forestry using eucalypts is of considerable importance to Paraguay, but knowledge regarding the pests and diseases affecting these plantations is limited. This study identified fungal diseases present in these plantations. Materials and Methods: We surveyed eucalypt plantations in four provinces in Paraguay and collected material from diseased trees for identification of the causal agents. The samples were analyzed using a combination of morphological and molecular methods. Results: Diseases encountered included Botryosphaeria stem canker, Calonectria leaf blight, Chrysoporthe stem canker, myrtle/eucalypt rust, Coniella leaf spot, heartwood rot and Teratosphaeria stem canker. Contrary to expectations, the causal agent of Teratosphaeria stem canker was identified as Teratosphaeria zuluensis (M.J. Wingf., Crous &amp, T.A. Cout.) M.J. Wingf. &amp, Crous and not Teratosphaeria gauchensis (M.-N. Cortinas, Crous &amp, M.J. Wingf.) M.J. Wingf. &amp, Crous, that is commonly documented for the South American region. Conclusions: This study updates the knowledge on forest fungal pathogens in Paraguayan eucalypt plantations and is the first report of T. zuluensis in Paraguay and in South America.

Root and butt rot caused by members of the Heterobasidion annosum species complex is the most economically important disease of conifer trees in boreal forests. Wood decay in the infected trees dramatically decreases their value and causes considerable losses to forest owners. Trees vary in their susceptibility to Heterobasidion infection, but the genetic determinants underlying the variation in the susceptibility are not well-understood. We performed the identification of Norway spruce genes associated with the resistance to Heterobasidion parviporum infection using genome-wide exon-capture approach. Sixty-four clonal Norway spruce lines were phenotyped, and their responses to H. parviporum inoculation were determined by lesion length measurements. Afterwards, the spruce lines were genotyped by targeted resequencing and identification of genetic variants (SNPs). Genome-wide association analysis identified 10 SNPs located within 8 genes as significantly associated with the larger necrotic lesions in response to H. parviporum inoculation. The genetic variants identified in our analysis are potential marker candidates for future screening programs aiming at the differentiation of disease-susceptible and resistant trees.

Plant microbiome plays an important role in maintaining the host fitness. Despite a significant progress in our understanding of the plant microbiome achieved in the recent years, very little is known about the effect of plant pathogens on composition of microbial communities associated with trees. In this study, we analysed the mycobiome of different anatomic parts of asymptomatic and symptomatic Norway spruce trees naturally infected by Heterobasidion spp. We also investigated the primary impact of the disease on the fungal communities, which are associated with Norway spruce trees. Our results demonstrate that the structure of fungal communities residing in the wood differed significantly among symptomatic and asymptomatic Heterobasidion-infected trees. However, no significant differences were found in the other anatomic regions of the trees. The results also show that not only each of individual tree tissues (wood, bark, needles and roots) harbours a unique fungal community, but also that symptomatic trees were more susceptible to co-infection by other wood-degrading fungi compared to the asymptomatic ones.

The successful implementation of next-generation sequencing techniques in plant and woody tissues depends on the quality of initial starting material. This study demonstrated the use of a modified protocol that enabled the simultaneous extraction of both genomic DNA and total RNA from recalcitrant woody material. The genetic material obtained by this protocol is of high quality and can be directly used in downstream analysis (e.g., next-generation sequencing). This protocol is particularly useful not only when the initial plant material is limited but also when genomic DNA features (e.g., methylation) have to be compared with the total RNA (e.g., gene expression). For such studies, the extraction from the same materials is highly preferred to minimize sample variation., Lay abstract The advancement of next-generation sequencing techniques has greatly facilitated almost all branches of biological and life-science studies. To guarantee reliable results and conclusions, high-quality starting genetic material (genomic DNA and total RNA) is essential. Our modified protocol, which is based on existing methods, enabled the efficient simultaneous extraction of genomic DNA and total RNA from the same recalcitrant woody sample. Thereby, it greatly minimizes the amount of initial plant materials needed for a specific study.

Symbiosis with microbes is crucial for survival and development of wood-inhabiting longhorn beetles (Coleoptera: Cerambycidae). Thus, knowledge of the endemic fungal associates of insects would facilitate risk assessment in cases where a new invasive pest occupies the same ecological niche. However, the diversity of fungi associated with insects remains poorly understood. The aim of this study was to investigate fungi associated with the native large poplar longhorn beetle (Saperda carcharias) and the recently introduced Asian longhorn beetle (Anoplophora glabripennis) infesting hardwood trees in Finland. We studied the cultivable fungal associates obtained from Populus tremula colonised by S. carcharias, and Betula pendula and Salix caprea infested by A. glabripennis, and compared these to the samples collected from intact wood material. This study detected a number of plant pathogenic and saprotrophic fungi, and species with known potential for enzymatic degradation of wood components. Phylogenetic analyses of the most commonly encountered fungi isolated from the longhorn beetles revealed an association with fungi residing in the Cadophora-Mollisia species complex. A commonly encountered fungus was Cadophora spadicis, a recently described fungus associated with wood-decay. In addition, a novel species of Cadophora, for which the name Cadophora margaritata sp. nov. is provided, was isolated from the colonised wood.

In the natural forest environment, the spread of pathogens may have dramatic effects on ecosystem functioning. To successfully control devastating forest pathogens, application of endophytes as biocontrol agents is an emerging area of research. There are several ways by which endophytic microorganisms can protect their tree hosts against pathogens. Endophytes promote plant growth by producing beneficial secondary metabolites (e.g. phytohormones) or providing nutrients (e.g. phosphorus). Endophytes can compete with pathogens and herbivores by successfully utilizing available substrates (colonization of shared niche can restrict pathogen invasion), or endophytes can produce antagonistic metabolites. Endophytes can enhance plant resistance by triggering and priming host defensive reactions. Endophytes could provide several opportunities for application in integrated pest management (IPM) to gain sustainable forestry practices. To utilize endophytes as biocontrol agents, the mechanisms behind the possible mode of action should be determined. Novel advances in cultivation-independent techniques including next generation sequencing technology (NGS), association analyses and network inference modelling will greatly facilitate identification of endophytes and unravel potential beneficial functions of endophytic communities. A further understanding of these mechanisms could help to minimize the use of environmental harming chemicals in plant and forest tree protection. We propose simple guidelines that could facilitate the use of fungal endophytes as biocontrol agents and simultaneously study their ecological functions.

Both the establishment of sustainable forestry practices and the improvement of commercially grown trees require better understanding of mechanisms used by forest trees to combat microbial pathogens. We investigated the contribution of a gene encoding Scots pine (Pinus sylvestris L.) antimicrobial protein Sp-AMP2 (PR-19) to the host defenses to evaluate the potential of Sp-AMP genes as molecular markers for resistance breeding. We developed transgenic tobacco plants expressing the Sp-AMP2 gene. Transgenic plants showed a reduction in the size of lesions caused by the necrotrophic pathogen Botrytis cinerea. In order to investigate Sp-AMP2 gene expression level, four transgenic lines were tested in comparison to control and non-transgenic plants. No Sp-AMP2 transcripts were observed in any of the control and non-transgenic plants tested. The transcript of Sp-AMP2 was abundantly present in all transgenic lines. Sp-AMP2 was induced highly in response to the B. cinerea infection at 3 d.p.i. This study provides an insight into the role of Sp-AMP2 and its functional and ecological significance in the regulation of plant–pathogen interactions.

Forest fires are a common natural disturbance in forested ecosystems and have a large impact on the microbial communities in forest soils. The response of soil fungal communities to forest fire is poorly documented. Here, we investigated fungal community structure and function across a 152-year boreal forest fire chronosequence using high-throughput sequencing of the internal transcribed spacer 2 (ITS2) region and a functional gene array (GeoChip). Our results demonstrate that the boreal forest soil fungal community was most diverse soon after a fire disturbance and declined over time. The differences in the fungal communities were explained by changes in the abundance of basidiomycetes and ascomycetes. Ectomycorrhizal (ECM) fungi contributed to the increase in basidiomycete abundance over time, with the operational taxonomic units (OTUs) representing the genera Cortinarius and Piloderma dominating in abundance. Hierarchical cluster analysis by using gene signal intensity revealed that the sites with different fire histories formed separate clusters, suggesting differences in the potential to maintain essential biogeochemical soil processes. The site with the greatest biological diversity had also the most diverse genes. The genes involved in organic matter degradation in the mature forest, in which ECM fungi were the most abundant, were as common in the youngest site, in which saprotrophic fungi had a relatively higher abundance. This study provides insight into the impact of fire disturbance on soil fungal community dynamics.

The cerato-platanin family is a group of small secreted cysteine-rich proteins exclusive for filamentous fungi. They have been shown to be involved in the interactions between fungi and plants. Functional characterization of members from this family has been performed mainly in Ascomycota, except Moniliophthora perniciosa. Our previous phylogenetic analysis revealed that recent gene duplication of cerato-platanins has occurred in Basidiomycota but not in Ascomycota, suggesting higher functional diversification of this protein family in Basidiomycota than in Ascomycota. In this study, we identified three cerato-platanin homologues from the basidiomycete conifer pathogen Heterobasidion annosum sensu stricto. Expression of the homologues under various conditions as well as their roles in the H. annosum s.s.-Pinus sylvestris (Scots pine) pathosystem was investigated. Results showed that HaCPL2 (cerato-platanin-like protein 2) had the highest sequence similarity to cerato-platanin from Ceratocystis platani and hacpl2 was significantly induced during nutrient starvation and necrotrophic growth. The treatment with recombinant HaCPL2 induced cell death, phytoalexin production and defense gene expression in Nicotiana tabacum. Eliciting and cell death-inducing ability accompanied by retardation of apical root growth was also demonstrated in Scots pine seedlings. Our results suggest that HaCPL2 might contribute to the virulence of H. annosum s.s. by promoting plant cell death.

The pine shoot beetle, Tomicus piniperda, is a secondary colonizer of pines and other conifers. Although it is necessary to understand interactions of this insect with other organisms, few studies have focused on its fungal associates. This study focused on the effect of geographical distance on the occurrence of fungi carried by the beetle. Adult beetles were collected from Scots pine (Pinus sylvestris) in North (Rovaniemi) and South (Hyytiala) Finland. The mycota was isolated and identified using a combination of morphological and molecular data. The results revealed a great diversity of fungal taxa isolated from T. piniperda, with a total of 3073 isolates representing 20 taxonomic units. The most frequently isolated fungi in the bark beetles from North Finland were Beauveria bassiana, Kuraishia sp., whereas Penicillium velutinum was mostly observed in the insects from South Finland. Ophiostoma canum and Ophiostoma minus were also isolated from the beetles from both North and South Finland. The number o...

Heterobasidion parviporum Niemelä & Korhonen is responsible for the majority of decay in conifers in northern Europe, which causes severe economic losses. In nature, heterokaryotic isolates of H. parviporum cause infection in Norway spruce (Picea abies (L.) Karst.). However, little is known on whether homokaryons of H. parviporum can infect trees under field conditions. In this study, 40-year-old clonal Norway spruce stems and roots were inoculated with a homokaryotic isolate of H. parviporum under field conditions. After four months, the infection frequency and necrotic lesion lengths were recorded. The homokaryon caused infection and provoked the development of necrotic lesions. Necrotic lesions were larger in roots than in stems. Among the studied Norway spruce genotypes, a Russian clone had the smallest necrotic lesions, whereas a Finnish clone developed the largest necrotic lesions. Clones with higher growth rates were more sensitive to fungal infection and wound damage. Under microscopic observation, H. parviporum grew adpressed to lumen cell walls, colonized tracheids next to rays, and induced lignification in cell walls close to the point of inoculation. This study provides a starting point for further studies on the ability of homokaryons to cause infection under field conditions and for discussions on factors affecting the resistance of Norway spruce against H. parviporum.

Background Heterobasidion parviporum is an economically most important fungal forest pathogen in northern Europe, causing root and butt rot disease of Norway spruce (Picea abies (L.) Karst.). The mechanisms underlying the pathogenesis and virulence of this species remain elusive. No reference genome to facilitate functional analysis is available for this species. Results To better understand the virulence factor at both phenotypic and genomic level, we characterized 15 H. parviporum isolates originating from different locations across Finland for virulence, vegetative growth, sporulation and saprotrophic wood decay. Wood decay capability and latitude of fungal origins exerted interactive effects on their virulence and appeared important for H. parviporum virulence. We sequenced the most virulent isolate, the first full genome sequences of H. parviporum as a reference genome, and re-sequenced the remaining 14 H. parviporum isolates. Genome-wide alignments and intrinsic polymorphism analysis showed that these isolates exhibited overall high genomic similarity with an average of at least 96% nucleotide identity when compared to the reference, yet had remarkable intra-specific level of polymorphism with a bias for CpG to TpG mutations. Reads mapping coverage analysis enabled the classification of all predicted genes into five groups and uncovered two genomic regions exclusively present in the reference with putative contribution to its higher virulence. Genes enriched for copy number variations (deletions and duplications) and nucleotide polymorphism were involved in oxidation-reduction processes and encoding domains relevant to transcription factors. Some secreted protein coding genes based on the genome-wide selection pressure, or the presence of variants were proposed as potential virulence candidates. Conclusion Our study reported on the first reference genome sequence for this Norway spruce pathogen (H. parviporum). Comparative genomics analysis gave insight into the overall genomic variation among this fungal species and also facilitated the identification of several secreted protein coding genes as putative virulence factors for the further functional analysis. We also analyzed and identified phenotypic traits potentially linked to its virulence. Electronic supplementary material The online version of this article (10.1186/s12864-018-4610-4) contains supplementary material, which is available to authorized users.

Norway spruce is one of the most important commercial forestry species in Europe, and is commonly infected by the bark beetle-vectored necrotrophic fungus, Endoconidiophora polonica. Spruce trees display a restricted capacity to respond to environmental perturbations, and we hypothesized that water limitation will increase disease severity in this pathosystem. To test this prediction, 737 seedlings were randomized to high (W+) or low (W−) water availability treatment groups, and experimentally inoculated with one of three E. polonica strains or mock-inoculated. Seedling mortality was monitored throughout an annual growing season, and total seedling growth and lesion length indices were measured at the experiment conclusion. Seedling growth was greater in the W+ than W− treatment group, demonstrating limitation due to water availability. For seedlings infected with two of the fungal strains, no differences in disease severity occurred in response to water availability. For the third fungal strain, however, greater disease severity (mortality and lesion lengths) occurred in W− than W+ seedlings. While the co-circulation in nature of multiple E. polonica strains of varying virulence is known, this is the first experimental evidence that water availability can alter strain-specific disease severity.

Silver birch (Betula pendula) is a pioneer boreal tree that can be induced to flower within 1 year. Its rapid life cycle, small (440-Mb) genome, and advanced germplasm resources make birch an attractive model for forest biotechnology. We assembled and chromosomally anchored the nuclear genome of an inbred B. pendula individual. Gene duplicates from the paleohexaploid event were enriched for transcriptional regulation, whereas tandem duplicates were overrepresented by environmental responses. Population resequencing of 80 individuals showed effective population size crashes at major points of climatic upheaval. Selective sweeps were enriched among polyploid duplicates encoding key developmental and physiological triggering functions, suggesting that local adaptation has tuned the timing of and cross-talk between fundamental plant processes. Variation around the tightly-linked light response genes PHYC and FRS10 correlated with latitude and longitude and temperature, and with precipitation for PHYC. Similar associations characterized the growth-promoting cytokinin response regulator ARR1, and the wood development genes KAK and MED5A., published version, peerReviewed

Climate changes, exemplified by increased temperatures and CO2 concentration, pose a global threat to forest health. Of particular concern are pests and pathogens, with a warming climate altering their distributions and evolutionary capacity, while impairing the ability of some plants to respond to infections. Progress in understanding and mitigating such effects is currently hindered by a lack of empirical research. Norway spruce (Picea abies) is one of the most economically important tree species in northern Europe, and is considered highly vulnerable to changes in climate. It is commonly infected by the fungus Endoconidiophora polonica, and we hypothesized that damage caused to trees will increase under future climate change predictions. To test this hypothesis an in vivo greenhouse experiment was conducted to evaluate the effects of a changed growing environment on E. polonica infected Norway spruce seedlings, comparing ambient conditions to predicted temperatures and CO2 levels in Finland for the years 2030 and 2100. In total, 450 seedlings were randomized amongst the three treatments, with 25 seedlings from each allocated to inoculation with one of five different fungal strains or mock-inoculation. Seedlings were monitored throughout the thermal growing season for mortality, and lesion length and depth indices were measured at the experiment conclusion. Disease severity (mortality and lesions) was consistently greater in fungal-inoculated than mock-inoculated seedlings. However, substantial differences were observed among fungal strains in response to climate scenarios. For example, although overall seedling mortality was highest under the most distant (and severe) climate change expectations, of the two fungal strains with the highest mortality counts (referred to as F4 and F5), one produced greater mortality under the 2030 and 2100 scenarios than ambient conditions, whereas climate scenario had no effect on the other. This study contributes to a limited body of empirical research on the effects of projected climate changes on forestry pathosystems, and is the first to investigate interactions between Norway spruce and E. polonica. The results indicate the potential for future climate changes to alter the impact of forest pathogens with implications for productivity, while highlighting the need for a strain-specific level of understanding of the disease agents.

NADPH oxidases (Noxes), transmembrane proteins found in most eukaryotic species, generate reactive oxygen species and are thereby involved in essential biological processes. However, the fact that genes encoding ferric reductases and ferric-chelate reductases share high sequence similarities and domains with Nox genes represents a challenge for bioinformatic approaches used to identify Nox-encoding genes. Further, most studies on fungal Nox genes have focused mainly on functionality, rather than sequence properties, and consequently clear differentiation among the various Nox isoforms has not been achieved. We conducted an extensive sequence analysis to identify putative Nox genes among 34 eukaryotes, including 28 fungal genomes and one Oomycota genome. Analyses were performed with respect to phylogeny, transmembrane helices, di-histidine distance and glycosylation. Our analyses indicate that the sequence properties of fungal Nox genes are different from those of human and plant Nox genes, thus providing novel insight that will enable more accurate identification and characterization of fungal Nox genes.

Most of the spruce-dominated pristine mires in South Finland have been drained for forestry, and a large proportion of these forest stands are currently approaching regeneration maturity. We investigated the potential impact of the drainage of pristine mire on the species composition of root endophytes compared to undisturbed mire and mineral soil. Additionally, we further evaluated the inhibitory effect of a subset of the isolated endophytes on the root rot pathogen Heterobasidion parviporum. A total of 113 isolates of fungal root endophytes were obtained from non-mycorrhizal Picea abies roots, which were assigned to 15 different OTUs. Most of the isolates consisted of dark septate endophytes (77 %); the Phialocephala fortinii s.l.–Acephala applanata species complex was the most dominant group, comprising 52 % of all the isolates. No significant differences in the frequency of isolates were observed between the sites. All the diversity indices suggest relatively low or mid-level versatility of the endophyte community, with mineral soil sites having the lowest index and drained peatland the highest. Nineteen of the isolates (17 %) inhibited the growth of the conifer root rot pathogen in vitro. The potential of these endophytes to serve as alternative antagonistic agents to control and manage Heterobasidion root rot disease is discussed.

Rigidoporus microporus (Polyporales, Basidiomycota) syn. Rigidoporus lignosus is the most destructive root pathogen of rubber plantations distributed in tropical and sub-tropical regions. Our primary objective was to characterize Nigerian isolates from rubber tree and compare them with other West African, Southeast Asian and American isolates. To characterize the 20 isolates from Nigeria, we used sequence data of the nuclear ribosomal DNA ITS and LSU, β-tubulin and translation elongation factor 1-α (tef1) gene sequences. Altogether, 40 isolates of R. microporus were included in the analyses. Isolates from Africa, Asia and South/Central America formed three distinctive clades corresponding to at least three species. No phylogeographic pattern was detected among R. microporus collected from West and Central African rubber plantations suggesting continuous gene flow among these populations. Our molecular phylogenetic analysis suggests the presence of two distinctive species associated with the white rot disease. Phylogenetic analyses placed R. microporus in the Hymenochaetales in the vicinity of Oxyporus. This is the first study to characterize R. microporus isolates from Nigeria through molecular phylogenetic techniques, and also the first to compare isolates from rubber plantations in Africa and Asia.

Bacteria in peat forest soil play important role in global carbon cycling. The distribution of bacteria population in different peat soils as a whole and how forest management practices alter the bacterial populations are still poorly known. Using pyrosequencing analysis of 16S rRNA gene, we quantified the diversity and community structure of bacteria in eight peat forest soils (pristine and drained) and two mineral forest soils from Lakkasuo, Finland with either spruce-dominant or pine-dominant tree species. In total, 191,229 sequences which ranged from 15,710 to 22,730 per sample were obtained and affiliated to 13 phyla, 30 classes and 155 genera. The peat forest soils showed high bacterial diversity and species richness. The tree species seems to have more strong impact on the bacterial diversity than the type of peat soil, which drives the changes in bacterial community structure. The dominant taxonomic groups across all soils (>1% of all sequences) were Proteobacteria , Acidobacteria , Actinobacteria , Bacteroidetes , Planctomycetes and Verrucomicrobia . The relative abundance of bacteria phylum and genus differed between soil types and between vegetation. Significant differences in relative abundance of bacteria phyla were only found for Gemmatimonadetes and Cyanobacteria between the pristine and the drained peat forest soils. At genus level, the relative abundance of several genera differed significantly between the peat soils with same or different tree species, including Burkholderia , Caulobacter , Opitutus , Mucilanginibacter , Acidocella , Mycobacterium , Bradyrhizobium , Dyella and Rhodanobacter .

Heterobasidion parviporum Niemel&auml, &amp, Korhonen is an economically important basidiomycete, causing root and stem rot disease of Norway spruce (Picea abies (L.) Karst) in Northern Europe. The H. parviporum genome encodes numerous small secreted proteins, which might be of importance for interacting with mycorrhiza symbionts, endophytes, and other saprotrophs. We hypothesized that small secreted proteins from H. parviporum (HpSSPs) are involved in interspecific fungal interaction. To identify HpSSP-coding genes potentially involved, we screened the H. parviporum effectome and compared their transcriptomic profiles during fungal development and in planta tree infection. We further conducted phylogenetic analysis, and identified a subset of hypothetical proteins with nonpredicted domain or unknown function as HpSSPs candidates for further characterization. The HpSSPs candidates were selected based on high-quality sequence, cysteine residue frequency, protein size, and in planta expression. We subsequently explored their roles during in vitro interaction in paired cultures of H. parviporum with ectomycorrhizal Cortinarius gentilis, endophytic Phialocephala sphaeroides, saprotrophs (Mycena sp., Phlebiopsis gigantea, and Phanerochaete chrysosporium), respectively. The transcriptomic profile revealed that a large proportion of effector candidates was either barely expressed or highly expressed under all growth conditions. In vitro dual-culture test showed that P. sphaeroides and C. gentilis were overgrown by H. parviporum. The barrage zone formation or no physical contact observed in paired cultures with the saprotrophs suggest they had either combative interaction or antibiosis effect with H. parviporum. Several HpSSPs individuals were up- or downregulated during the nonself interactions. The results of HpSSPs gene expression patterns provide additional insights into the diverse roles of SSPs in tree infection and interspecific fungal interactions.

Conifer trees, including Norway spruce, are threatened by fungi of the Heterobasidion annosum species complex, which severely affect timber quality and cause economic losses to forest owners. The timely detection of infected trees is complicated, as the pathogen resides within the heartwood and sapwood of infected trees. The presence of the disease and the extent of the wood decay often becomes evident only after tree felling. Fourier-transform infrared (FT-IR) spectroscopy is a potential method for non-destructive sample analysis that may be useful for identifying infected trees in this pathosystem. We performed FT-IR analysis of 18 phloem, 18 xylem, and 18 needle samples from asymptomatic and symptomatic Norway spruce trees. FT-IR spectra from 1066 – 912 cm−1 could be used to distinguish phloem, xylem, and needle tissue extracts. FT-IR spectra collected from xylem and needle extracts could also be used to discriminate between asymptomatic and symptomatic trees using spectral bands from 1657 – 994 cm−1 and 1104 – 994 cm−1, respectively. A partial least squares regression model predicted the concentration of condensed tannins, a defense-related compound, in phloem of asymptomatic and symptomatic trees. This work is the first to show that FT-IR spectroscopy can be used for the identification of Norway spruce trees naturally infected with Heterobasidion spp.

Terrestrial plants including forest trees are generally known to live in close association with microbial organisms. The inherent features of this close association can be commensalism, parasitism or mutualism. The term “microbiota” has been used to describe this ecological community of plant-associated pathogenic, mutualistic, endophytic and commensal microorganisms. Many of these microbiota inhabiting forest trees could have a potential impact on the health of, and disease progression in, forest biomes. Comparatively, studies on forest tree microbiomes and their roles in mutualism and disease lag far behind parallel work on crop and human microbiome projects. Very recently, our understanding of plant and tree microbiomes has been enriched due to novel technological advances using metabarcoding, metagenomics, metatranscriptomics and metaproteomics approaches. In addition, the availability of massive DNA databases (e.g., NCBI (USA), EMBL (Europe), DDBJ (Japan), UNITE (Estonia)) as well as powerful computational and bioinformatics tools has helped to facilitate data mining by researchers across diverse disciplines. Available data demonstrate that plant phyllosphere bacterial communities are dominated by members of only a few phyla (Proteobacteria, Actinobacteria, Bacteroidetes). In bulk forest soil, the dominant fungal group is Basidiomycota, whereas Ascomycota is the most prevalent group within plant tissues. The current challenge, however, is how to harness and link the acquired knowledge on microbiomes for translational forest management. Among tree-associated microorganisms, endophytic fungal biota are attracting a lot of attention for their beneficial health- and growth-promoting effects, and were preferentially discussed in this review.

Heterobasidion annosum is widely known as a major root and butt rot pathogen of conifer trees, but little information is available on its interaction with the roots of herbaceous angiosperm plants. We investigated the infection biology of H. annosum during challenge with the angiosperm model Arabidopsis and monitored the host response after exposure to different hormone elicitors, chemicals (chitin, glucan and chitosan) and fungal species that represent diverse basidiomycete life strategies [e.g., pathogen (H. annosum), saprotroph (Stereum sanguinolentum) and mutualist (Lactarius rufus)]. The results revealed that the tree pathogen (H. annosum) and the saprotroph (S. sanguinolentum) could infect the Col-8 (Columbia) ecotype of Arabidopsis in laboratory inoculation experiments. Germinated H. annosum spores had appressorium-like penetration structures attached to the surface of the Arabidopsis roots. Subsequent invasive fungal growth led to the disintegration of the vascular region of the root tissues. Progression of root rot symptoms in Arabidopsis was similar to the infection development that was previously documented in Scots pine seedlings. Scots pine PsDef1 and Arabidopsis DEFLs (AT5G44973.1) and PDF1.2 were induced at the initial stage of the infection. However, differences in the expression patterns of the defensin gene homologs from the two plant groups were observed under various conditions, suggesting functional differences in their regulation. The potential use of the H. annosum-Arabidopsis pathosystem as a model for studying forest tree diseases is discussed.

Summary The success of many wood decaying fungi lies in their ability to overcome unfavourable environmental conditions within and outside of litter and wood debris. Although so much has been learned about the ecology, taxonomy and physiology of several wood decaying basidiomycete fungi, the molecular basis for their survival in a diverse range of substrates and ecological habitats has been very little studied. Using the wood decay fungus (Heterobasidion annosum s.s.) as a model, we investigated its transcriptomic response when exposed to several environmental stressors (high and low temperature, osmotic stress, oxidative stress and nutrient starvation) and during growth on specific pine wood compartments (bark, sapwood and heartwood). Among other genes and pathways, we documented the specific induction of the major facilitator superfamily 1 and cytochrome P450 families at low temperature, and protein kinases together with transcription factors during starvation. On the other hand, during saprotrophic growth, we observed the induction of many glycosyl hydrolases, three multi-copper oxidases (MCO), five manganese peroxidases (MnP) and one oxidoreductase which are specific for wood degradation. This is the first study providing insights on the potential mechanisms for adaptation to abiotic stresses and pine heartwood degradation in H. annosum s.s.

The process of mating in Basidiomycota is regulated by homeodomain-encoding genes (HD) and pheromones and G protein-coupled pheromone receptor genes (P/R). Whether these genes are actually involved in determining mating type distinguishes mating systems that are considered tetrapolar (two locus) from bipolar (one locus). Polyporales are a diverse group of wood-decay basidiomycetes displaying high variability in mating and decay systems. Many of the bipolar species appear to be brown-rot fungi, and it has been hypothesized that there is a functional basis for this correlation. Here we characterize mating genes in recently sequenced Polyporales and other Agaricomycete genomes. All Agaricomycete genomes encode HD and pheromone receptor genes regardless of whether they are bipolar or tetrapolar. The HD genes are organized into a MAT-HD locus with a high degree of gene order conservation among neighboring genes, with the gene encoding mitochondrial intermediate peptidase consistently syntenic but no linkage to the P/R genes. To have a complete dataset of species with known mating systems we determined that Wolfiporia cocos appears to be bipolar, using the criterion that DNA polymorphism of MAT genes should be extreme. Testing the correlation of mating and decay systems while controlling for phylogenetic relatedness failed to identify a statistical association, likely due to the small number of taxa employed. Using a phylogenetic analysis of Ste3 proteins, we identified clades of sequences that contain no known mating type-specific receptors and therefore might have evolved novel functions. The data are consistent with multiple origins of bipolarity within the Agaricomycetes and Polyporales, although the alternative hypothesis that tetrapolarity and bipolarity are reversible states needs better testing.

The biocontrol agent Phlebiopsis gigantea has been intensively applied to the surface of Picea abies stumps to control Heterobasidion root rot. But little is known about the possible impact of this treatment on the resident bacteria community in the stumps. High throughput DNA bar-coded pyrosequencing was used to characterize the diversity of bacteria in the stumps of P. abies at 1, 6 and 13 years after treatment with P. gigantea. The sequences were classified into 12 phyla and 160 genera, of which Proteobacteria and Acidobacteria were the most abundant groups over time. Moreover, at the initial stages of decay, Proteobacteria were the most abundant whereas Acidobacteria were the most common at advanced stages of decay. Treatment with P. gigantea led to significant increase of the genus Acidobacteria-Gp1 at 1 year after treatment. The analysis of observed and estimated operational taxonomic units (OTUs) as well as diversity indices revealed that P. gigantea treatment significantly decreased the initial bacterial richness in the stumps, but the bacterial community gradually recovered and the negative effects of P. gigantea was attenuated. These results provide additional insight on the risk assessment as well as environmental impact on the long-term use of P. gigantea in the control of Heterobasidion root rot in conifer forests.

Climate changes, exemplified by increased temperatures and CO2 concentration, pose a global threat to forest health. Of particular concern are pests and pathogens, with a warming climate altering their distributions and evolutionary capacity, while impairing the ability of some plants to respond to infections. Progress in understanding and mitigating such effects is currently hindered by a lack of empirical research. Norway spruce (Picea abies) is one of the most economically important tree species in northern Europe, and is considered highly vulnerable to changes in climate. It is commonly infected by the fungus Endoconidiophora polonica, and we hypothesized that damage caused to trees will increase under future climate change predictions. To test this hypothesis an in vivo greenhouse experiment was conducted to evaluate the effects of a changed growing environment on E. polonica infected Norway spruce seedlings, comparing ambient conditions to predicted temperatures and CO2 levels in Finland for the years 2030 and 2100. In total, 450 seedlings were randomized amongst the three treatments, with 25 seedlings from each allocated to inoculation with one of five different fungal strains or mock-inoculation. Seedlings were monitored throughout the thermal growing season for mortality, and lesion length and depth indices were measured at the experiment conclusion. Disease severity (mortality and lesions) was consistently greater in fungal-inoculated than mock-inoculated seedlings. However, substantial differences were observed among fungal strains in response to climate scenarios. For example, although overall seedling mortality was highest under the most distant (and severe) climate change expectations, of the two fungal strains with the highest mortality counts (referred to as F4 and F5), one produced greater mortality under the 2030 and 2100 scenarios than ambient conditions, whereas climate scenario had no effect on the other. This study contributes to a limited body of empirical research on the effects of projected climate changes on forestry pathosystems, and is the first to investigate interactions between Norway spruce and E. polonica. The results indicate the potential for future climate changes to alter the impact of forest pathogens with implications for productivity, while highlighting the need for a strain-specific level of understanding of the disease agents.

Boreal peatlands play a crucial role in global carbon cycling, acting as an important carbon reservoir. However, little information is available on how peatland microbial communities are influenced by natural variability or human-induced disturbances. In this study, we have investigated the fungal diversity and community structure of both the organic soil layer and buried wood in boreal forest soils using high-throughput sequencing of the internal transcribed spacer (ITS) region. We have also compared the fungal communities during the primary colonization of wood with those of the surrounding soils. A permutational multivariate analysis of variance (PERMANOVA) confirmed that the community composition significantly differed between soil types ( P < 0.001) and tree species ( P < 0.001). The distance-based linear models analysis showed that environmental variables were significantly correlated with community structure ( P < 0.04). The availability of soil nutrients (Ca [ P = 0.002], Fe [ P = 0.003], and P [ P = 0.003]) within the site was an important factor in the fungal community composition. The species richness in wood was significantly lower than in the corresponding soil ( P < 0.004). The results of the molecular identification were supplemented by fruiting body surveys. Seven of the genera of Agaricomycotina identified in our surveys were among the top 20 genera observed in pyrosequencing data. Our study is the first, to our knowledge, fungal high-throughput next-generation sequencing study performed on peatlands; it further provides a baseline for the investigation of the dynamics of the fungal community in the boreal peatlands.

Soil microbial responses to fire are likely to change over the course of forest recovery. Investigations on long-term changes in bacterial dynamics following fire are rare. We characterized the soil bacterial communities across three different times post fire in a 2 to 152-year fire chronosequence by Illumina MiSeq sequencing, coupled with a functional gene array (GeoChip). The results showed that the bacterial diversity did not differ between the recently and older burned areas, suggesting a concomitant recovery in the bacterial diversity after fire. The differences in bacterial communities over time were mainly driven by the rare operational taxonomic units (OTUs, published version, peerReviewed

A key tree species for the forest industry in Europe is Norway spruce [Picea abies (L.) Karst.]. One of its major diseases is stem and butt rot caused by Heterobasidion parviporum (Fr.) Niemela & Korhonen, which causes extensive revenue losses every year. In this study, we investigated the parallel induction of Norway spruce genes presumably associated with salicylic acid- and jasmonic acid/ethylene-mediated signalling pathways previously observed in response to H. parviporum. Relative gene expression levels in bark samples of genes involved in the salicylic acid- and jasmonic acid/ethylene-mediated signalling pathways after wounding and inoculation with either the saprotrophic biocontrol fungus Phlebiopsis gigantea or with H. parviporum were analysed with quantitative PCR at the site of the wound and at two distal locations from the wound/inoculation site to evaluate their roles in the induced defence response to H. parviporum in Norway spruce. Treatment of Norway spruce seedlings with methylsalicylate, methyljasmonate and inhibitors of the jasmonic acid/ethylene signalling pathway, as well as the Phenylalanine ammonia lyase inhibitor 2-aminoindan-2-phosphonic acid were conducted to determine the responsiveness of genes characteristic of the different pathways to different hormonal stimuli. The data suggest that jasmonic acid-mediated signalling plays a central role in the induction of the genes analysed in this study irrespective of their responsiveness to salicylic acid. This may suggest that jasmonic acid-mediated signalling is the prioritized module in the Norway spruce defence signalling network against H. parviporum and that there seems to be no immediate antagonism between the modules in this interaction.

Phlebiopsis gigantea has been widely used as the biocontrol fungus against the root and butt rot disease of conifers caused by Heterobasidion annosum. We investigated the regulation of two hydrophobin genes (Pgh1 and Pgh2) in strong and weak antagonistic isolates of the biological control agent P. gigantea under diverse substrate conditions. Transcript abundance of Pgh1 was higher in single cultures of strong performing isolates than in the weak performing isolates at the early and late stages of the fungal growth (P =0.05). Higher fold transcript changes of Pgh1 and Pgh2 were observed in the strong performing isolates at the early stage of the antagonistic interaction on modified Norkrans sawdust agar medium compared to the weak performing isolates. Higher transcript abundance of the two genes was also observed during growth in submerged compared to surface agar cultures (P